This invention relates generally to devices for carrying out biological analyses, particularly in vitro processes involving living cells such as cell culture and subsequent assays evaluating cell function. More particularly, this invention relates to multi-welled cell culture plates with permeable membranes sealed to the bottom of each of the wells.
The technology explosion associated with the biotech industry has seen a concomitant growth in the "biotechware" industry, where the need for a variety of dishes, flasks, tubes and plates to support biological studies is paramount. In many experiments involving biomolecules there is a stringent requirement to maintain sterile conditions as much as possible. This is particularly the case in carrying out in vitro processes involving living cells.
Devices used in conjunction with the in vitro growth and subsequent analysis of living cells are available from a number of manufacturers such as A/S Nunc and Corning. Such devices are available in various forms ranging from large plastic flasks, to which media and/or reagents are added to culture or assay the living cells, to plates having a plurality of self-contained impermeable plastic wells (usually available in 6-, 12- or 24-well versions), in which cell culture media or biological reagents are added to each individual well.
It has been shown that benefits ensue when cells are grown and studied with the aid of a permeable microporous membrane suspended in the cell growth medium. The microporous membrane usually is sealed to one end of a plastic cylinder which then is placed into a well of a culture plate with culture medium. Cells are placed in the chamber above and sometimes below the membrane. The microporous membrane allows free diffusion of ions and molecules so that cells more closely resemble their in vivo state than when grown on solid, impermeable plastic surfaces. The membrane allows liquid access to both sides of the cell, thereby improving cell differentiation and facilitating studies of cell transport and permeability as well as cell-cell interactions. The Millicell.RTM. culture plate insert sold by Millipore Corporation is an example of such a membrane device.
More recently, the concept of providing membrane wells for cell studies has been expanded to provide multiple-well plates having an array of wells with open bottoms and a microporous membrane sealed to the bottom of each well (hereinafter referred to as a "microwell membrane plate"). The microwell membrane plate is adapted to be inserted into a tray having a mating pattern of closed-end wells. Tissue culture media and/or reagents can be provided above and below the membranes, and cells can be added as desired to perform multiple studies simultaneously. The filter plate element of a MultiScreen.RTM. plate sold by Millipore Corporation is an example of such a microwell membrane plate.
Often it is desired to grow cells on the surface of the microporous membranes for several days before undertaking cell biology studies in order to allow attachment dependent cells to form a confluent layer on the membrane and to express their fully differentiated anatomical and physiological functions. A multi-welled tray of the type described above having mating closed-end wells can be used with a microwell membrane plate during the cell growth period; however, the culture media must be changed often to replenish nutrients used by the growing cells. It is advantageous during this initial growth period to use a tray with a few large reservoirs or even a single large reservoir which accepts all the filter wells of the microwell membrane plate. The large tray reservoirs provide a greater volume of culture medium per well than can a reservoir which accepts only one filter well, thus the medium does not have to be changed as often. Also, it is easier to change the culture medium in a single large reservoir rather than to change it in as many as 96 small reservoirs per tray.
Regardless of the type of device employed, experimentation involving living cells requires attention to sterility. Microbiological contamination is always an important concern when handling living cells. However, in this regard, it is not possible to hermetically seal the plates housing the growing cells from the ambient environment because an exchange of gas from the cells to the external environment and vice-versa is necessary. Thus covers which are used with such plates must provide a space between the cover and the container housing the cells. During handling of these cell analysis devices, the liquid solutions employed may spill and remain in the space between the cover and the container. This provides a pathway for contamination to enter the container.
The foregoing problem associated with spillage becomes magnified when dealing with a microwell membrane plate, such as the aforementioned MultiScreen.RTM. device, when used with a tray which has a single reservoir for media to interface with all cell-containing wells through the membrane. In this instance, the tray reservoir is relatively large and is filled almost to its rim with liquid. Since the tray cannot be sealed to the plate (or a cover for the plate), spillage is likely when the tray and plate are tilted or jostled during handling. Thus liquid will readily seep into the space between the tray and plate and remain there, thereby creating a contamination pathway which could adversely effect the cells or any subsequent analysis performed thereon.